Effects of Antiplatelet Drugs on Platelet-Dependent Coagulation Reactions.

Activated platelets are involved in blood coagulation by exposing phosphatidylserine (PS), which serves as a substrate for assembling coagulation complexes. Platelets accelerate fibrin formation and thrombin generation, two final reactions of the coagulation cascade. We investigated the effects of antiplatelet drugs on platelet impact in these reactions and platelet ability to expose PS. Washed human platelets were incubated with acetylsalicylic acid (ASA), ticagrelor, ASA in combination with ticagrelor, ruciromab (glycoprotein IIb-IIIa antagonist), or prostaglandin E1 (PGE1). Platelets were not activated or activated by collagen and sedimented in multiwell plates, and plasma was added after supernatant removal. Fibrin formation (clotting) was monitored in a recalcification assay by light absorbance and thrombin generation in a fluorogenic test. PS exposure was assessed by annexin V staining using flow cytometry. Ticagrelor (alone and in combination with ASA), ruciromab, and PGE1, but not ASA, prolonged the lag phase and decreased the maximum rate of plasma clotting and decreased the peak and maximum rate of thrombin generation. Inhibition was observed when platelets were not treated with exogenous agonists (activation by endogenous thrombin) and pretreated with collagen. Ticagrelor (alone and in combination with ASA), ruciromab, and PGE1, but not ASA, decreased PS exposure on washed platelets activated by thrombin and by thrombin + collagen. PS exposure on activated platelets in whole blood was lower in patients with acute coronary syndrome receiving ticagrelor + ASA in comparison with donors free of medications. These results indicate that antiplatelet drugs are able to suppress platelet coagulation activity not only in vitro but also after administration to patients.

Effects of platelets activated by different agonists on fibrin formation and thrombin generation.

Activated platelets possess procoagulant activity expressing on their surface phosphatidylserine (PS), a substrate for assembling coagulation complexes. We examined the effects of platelets activated by different agonists on fibrin formation and thrombin generation and compared these effects with each other and with PS expression. Modified plasma recalcification assay was developed to assess platelet effects on fibrin formation. Washed human platelets were left intact or activated by A23187 ionophore, collagen, arachidonic acid, ADP or TRAP (Thrombin Receptor Activating Peptide) and spun down in 96-well plates. Plasma was then added, recalcified, and fibrin formation was monitored by light absorbance. Platelets prepared in the same way were tested for their effect on thrombin generation. PS expression was evaluated by flow cytometry using annexin V staining. Platelets significantly accelerated fibrin formation and thrombin generation. They shortened lag phase and increased maximum rate of plasma clotting, and increased peak and maximum rate of thrombin generation. In both tests platelets were presumably activated by endogenous thrombin formed in plasma after triggering coagulation reactions. However, pretreatment with exogenous agonists additionally increased platelet procoagulant activity. It reached the maximum after incubation with A23187, being lower with collagen and arachidonic acid and minimum with ADP and TRAP (the latter might be ineffective due to competition with endogenous thrombin). The effects of platelets activated by different agonists on fibrin formation and thrombin generation correlate with each other and correspond to PS expression on their surface.

Why was the study done? Platelets and blood coagulation system interact with each other in hemostasis and intravascular thrombosis.Direct platelet effects on fibrin formation (plasma clotting), the final stage of blood coagulation cascade, have been insufficiently studied.The work is aimed at developing a method for studying platelet participation in fibrin formation in blood plasma and investigating the influence of platelet agonists on this reaction.What is new? Platelets significantly accelerate fibrin formation and their activation with various agonists (thrombin, collagen, arachidonic acid) enhances these effects.Effects of platelets on fibrin formation correlated with their ability to stimulate thrombin generation in blood plasmaEffects of platelets on fibrin formation and thrombin generation correlated with the level of phosphatidylserine exposure on their surfaceWhat is the impact? This study provides further evidence that platelet procоagulant effects on fibrin formation should be considered in investigations of platelet involvement in hemostatic and thrombotic reactions and in the evaluation of the efficacy of antiplatelet drugs.

CYP2C9 and VKORC1 genotyping for the quality of long-standing warfarin treatment in Russian patients.

A total of 263 warfarin naive patients with indications to long-term anticoagulation were included in prospective multicenter study and randomized into Pharmacogenetics and Standard dosing groups. The loading warfarin dose in Pharmacogenetics group was calculated by Gage algorithm and corrected starting on day 5 of treatment according to INR. In Standard dosing group warfarin initial dose was 5 mg and starting on day 3 of treatment it was titrated according to INR. Pharmacogenetics dosing in comparison with prescription of starting dose of 5 mg decreased major bleedings (0 vs. 6, p = 0.031), time to target INR (11 [9-14] vs. 17 [15-24] days, p = 0.046), and frequency of INR fluctuations ≥4.0 (11% vs. 30.9%, p = 0.002). The advantages of the pharmacogenetics dosing were mainly achieved due to the patients with increased warfarin sensitivity.

Plasma urokinase antigen and C-reactive protein predict angina recurrence after coronary angioplasty.

This study evaluates the predictive value of several biochemical indices of the coagulation-fibrinolysis system, platelet function, and inflammatory state for angina recurrence after successful percutaneous transluminal coronary angioplasty (PTCA). We measured preprocedural and follow-up plasma levels of C-reactive protein (CRP), fibrinogen, and urokinase plasminogen activator antigen (uPA), plasminogen activator inhibitor-1 (PAI-1) activity, tissue plasminogen activator activity, and adenosine diphosphate-induced platelet aggregation in 53 patients with chronic stable angina who underwent successful elective PTCA of single hemodynamically significant lesions in coronary arteries. All patients were followed up for 12 months after PTCA. The Cox proportional hazards model was used to assess the association of variables with angina recurrence rate. At the end of the follow-up, 16 patients had angina recurrence. Among 36 clinical, biochemical, and angiographic variables, the duration of stable angina more than 12 months before PTCA (χ (2) = 5.73; P = 0.02, hazard ratio (HR) 3.7, 95 % confidence interval (CI) 1.26-10.6), high baseline levels of CRP (>7 mg/l) (χ (2) = 8.34; P = 0.004, HR 2.9, 95 % CI 1.4-5.9), uPA antigen baseline (>1 ng/ml) (χ (2) = 17.11; P = 0.0001, HR 11.5, 95 % CI 3.6-36.7) and 48 h after PTCA (χ (2) = 15.73; P = 0.0001, HR 8.8, 95 % CI 3.01-25.96), baseline PAI-1 activity (>18 IU/ml) (χ (2) = 9.37; P = 0.002, HR 7.6, 95 % CI 2.07-27.84) were significant predictors of recurrent angina by univariate analyses. According to stepwise multivariate analyses, only the levels of plasma uPA antigen and serum CRP were shown to be significant independent predictors of angina recurrence (multivariate uPA χ (2) = 8.22, P = 0.004, HR 6.2, 95 % CI 1.78-21.67; CRP χ (2) = 4.09, P = 0.04, HR 1.9, 95 % CI 1.02-3.68). High preprocedural plasma uPA and serum CRP levels are indicative of angina recurrence after successful PTCA, and are valuable for the prognosis of restenosis.